Post on 01-Oct-2021
DRK-Blutspendedienst NSTOB - Institut Springe
The two RH genes and their
Rhesus boxes
FF Wagner
Priv.-Doz. Dr. Franz F Wagner
Abt. Spenderlabor
Institut Springe
31830 Springe
2DRK-Blutspendedienst NSTOB - Institut Springe
RH: The most complex blood group
� Immunohematology
�48 different antigens
�Antigen D split in >30 different epitopes
� Genetics
�>100 known haplotypes
�Similar phenotypes of different alleles
� Proteomics
�Membrane expression depends on RhAG
�Direct binding to Ankyrin
3DRK-Blutspendedienst NSTOB - Institut Springe
RH Blood Group
� Rh protein structure
�Structural model
�Differences of RhD and RhCE
� Genetics
�Structure of an RH gene
�Structure of RH locus and the RHD deletion
� Prediction of the phenotype
�Molecular basis of antigen D
�Molecular basis of other main antigens
�Testing strategies, pitfalls and limitations
4DRK-Blutspendedienst NSTOB - Institut Springe
RH Blood Group
� Rh protein structure
�Structural model
�Differences of RhD and RhCE
� Genetics
�Structure of an RH gene
�Structure of RH locus and the RHD deletion
� Prediction of the phenotype
�Molecular basis of antigen D
�Molecular basis of other main antigens
�Testing strategies, pitfalls and limitations
5DRK-Blutspendedienst NSTOB - Institut Springe
Rh protein in the membrane
� Complex with RhAG
� Direct interaction with ankyrin
Nicolas JBC 2003
6DRK-Blutspendedienst NSTOB - Institut Springe
12 transmembraneous helices
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Schematic structure of Rh protein
6 exofacial loops
7DRK-Blutspendedienst NSTOB - Institut Springe
Added complexity: AmtB structure resolved
� AmtB:
Ammonia transporter
of E. coli
� Homology to the Rh
proteins
� 3D structure resolved:
�3 protein complex
�11 helicesKhademi et al. Science 2004; 305: 1587
8DRK-Blutspendedienst NSTOB - Institut Springe
AmtB
� extracellular aminoterminal end
� helix 1 near the axis
Khademi et al. Science 2004; 305: 1587
9DRK-Blutspendedienst NSTOB - Institut Springe
RhD structure modeled on AmtB
32
49 52 131 135 186 201 263 266 321 324 391
417
35 80 107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
10DRK-Blutspendedienst NSTOB - Institut Springe
RhD structure modeled on AmtB
32
49 52 131 135 186 201 263 266 321 324 391
417
35 80 107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
Mature proteinstarts at position 2
11DRK-Blutspendedienst NSTOB - Institut Springe
RhD structure modeled on AmtB
32
49 52 131 135 186 201 263 266 321 324 391
417
35 80 107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
Mature proteinstarts at position 2
N-terminal endintracellular
12DRK-Blutspendedienst NSTOB - Institut Springe
RhD structure modeled on AmtB
32
49 52 131 135 186 201 263 266 321 324 391
417
35 80 107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
Mature proteinstarts at position 2
N-terminal endintracellular
Trypsin site42/43
13DRK-Blutspendedienst NSTOB - Institut Springe
RhD structure modeled on AmtB
32
49 52 131 135 186 201 263 266 321 324 391
417
35 80 107 158 167 230 231 282 290 347 358
Intracellular
Mature proteinstarts at position 2
N-terminal endintracellular
Trypsin site42/43
The AmtB model seems to fail at the aminoterminal end.
14DRK-Blutspendedienst NSTOB - Institut Springe
RhD structure partially modeled on AmtB
131 135 186 201 263 266 321 324 391
417
107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
32
11
2
72
53
75
94
15DRK-Blutspendedienst NSTOB - Institut Springe
RhD structure and AmtB
� Amino acids lining Ammonia channel
� 103 Phe
� 107 Phe
� 148 Trp
� 219 Ser
� 168 His
� 318 His
16DRK-Blutspendedienst NSTOB - Institut Springe
RhD structure and AmtB
� Amino acids lining Ammonia channel
� 103 Phe
� 107 Phe conserved through RhAG, RhBG, RhCG
� 148 Trp
� 219 Ser
� 168 His conserved through RhAG, RhBG, RhCG
� 318 His conserved through RhAG, RhBG, RhCG
17DRK-Blutspendedienst NSTOB - Institut Springe
RhD structure and AmtB
� Amino acids lining Ammonia channel
� 103 Phe not conserved in RhCE and RhD
� 107 Phe not conserved in RhCE and RhD
� 148 Trp not conserved in RhCE and RhD
� 219 Ser not conserved in RhCE and RhD
� 168 His not conserved in RhCE and RhD
� 318 His not conserved in RhCE and RhD
18DRK-Blutspendedienst NSTOB - Institut Springe
RH Blood Group
� Rh protein structure
�Structural model
�Differences of RhD and RhCE
� Genetics
�Structure of an RH gene
�Structure of RH locus and the RHD deletion
� Prediction of the phenotype
�Molecular basis of antigen D
�Molecular basis of other main antigens
�Testing strategies, pitfalls and limitations
19DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
RhD vs RhCE
20DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
RhD vs RhCE
21DRK-Blutspendedienst NSTOB - Institut Springe
RhD vs RhCE (new model)
131 135 186 201 263 266 321 324 391
417
107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
32
11
2
72
53
75
94
22DRK-Blutspendedienst NSTOB - Institut Springe
RhD vs RhCE (new model)
131 135 186 201 263 266 321 324 391
417
107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
32
11
2
72
53
75
94
23DRK-Blutspendedienst NSTOB - Institut Springe
RhD vs RhCE (new model)
131 135 186 201 263 266 321 324 391
417
107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
32
11
2
72
53
75
94
24DRK-Blutspendedienst NSTOB - Institut Springe
RH Blood Group
� Rh protein structure
�Structural model
�Differences of RhD and RhCE
� Genetics
�Structure of an RH gene
�Structure of RH locus and the RHD deletion
� Prediction of the phenotype
�Molecular basis of antigen D
�Molecular basis of other main antigens
�Testing strategies, pitfalls and limitations
25DRK-Blutspendedienst NSTOB - Institut Springe
Genomic structure
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Intracellular
Exofacial
131 135 186 201 263 266 321 324 391
417
107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
32
11
2
72
53
75
94
Partial deletion of intron 4 in RHDInsertion in intron 2 of C
26DRK-Blutspendedienst NSTOB - Institut Springe
RH Blood Group
� Rh protein structure
�Structural model
�Differences of RhD and RhCE
� Genetics
�Structure of an RH gene
�Structure of RH locus and the RHD deletion
� Prediction of the phenotype
�Molecular basis of antigen D
�Molecular basis of other main antigens
�Testing strategies, pitfalls and limitations
27DRK-Blutspendedienst NSTOB - Institut Springe
RH locus
Ancestral (Mouse)
P N RHSMP1
P N RHD
Hybrid
RHCESMP1
Rhesus box
Downstream
RHCESMP1
Upstream
Man
RHD positive
RHD negativeP N
50,000 bp
28DRK-Blutspendedienst NSTOB - Institut Springe
Detection of RHD deletion by PCR
cd
e/c
de
Cd
e/C
de
cd
E/c
dE
CD
e/c
de
cD
e/c
de
cD
E/c
de
CD
e/C
De
CD
e/c
DE
cD
E/c
DE
9,416 bp
6,557 bp
23,130 bp
RHD SMP1
SMP1
RHCE
RHCE
5’
5’
29DRK-Blutspendedienst NSTOB - Institut Springe
Detection of RHD deletion by PCR
PstI Sequence-specific
Priming
Hybrid Rhesus box
Upstream Rhesus box
Downstream Rhesus box
744 bp
100 bp
800 bp
179 bp
397 bp
564 bp
1,888 bp
30DRK-Blutspendedienst NSTOB - Institut Springe
Detection of RHD deletion by PCR
PstI Sequence-specific
Priming
Hybrid Rhesus box
Upstream Rhesus box
Downstream Rhesus box
Upstream
(RHD psi, DAU-1, -3)
Rhesus box
Downstream
(Weak D type 4.1)
Rhesus box
31DRK-Blutspendedienst NSTOB - Institut Springe
RH locus
RHD RHCESMP1A
B
RHD-CE(4-7)-D RHCE
B
C
B
RHD(1-9)-CE(10) RHCE(1)-D(2-10)
D
E
32DRK-Blutspendedienst NSTOB - Institut Springe
RH Blood Group
� Rh protein structure
�Structural model
�Differences of RhD and RhCE
� Genetics
�Structure of an RH gene
�Structure of RH locus and the RHD deletion
� Prediction of the phenotype
�Molecular basis of antigen D
�Molecular basis of other main antigens
�Testing strategies, pitfalls and limitations
33DRK-Blutspendedienst NSTOB - Institut Springe
Three types of D neg
RHD SMP1
SMP1
RHCE
RHCE
5’
5’
RHD SMP1 RHCE5’
RHD SMP1 RHCE5’
D+
D-
D-
D-
RHD deletion
„large“ hybrid
Stop codon, frameshift, splice site mutation ...
34DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen D
35DRK-Blutspendedienst NSTOB - Institut Springe
RhD loops and D antigen
D cat VD DBT CE
DFR D cat VI
D cat IV DHAR
Rh33Evans
Rh32DW
BARCFPTT
6
4 3
36DRK-Blutspendedienst NSTOB - Institut Springe
D negative RHD-CE-D hybrid alleles
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Intracellular
Exofacial
131 135 186 201 263 266 321 324 391
417
107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
32
11
2
72
53
75
94
Partial deletion of intron 4 in RHDInsertion in intron 2 of C
37DRK-Blutspendedienst NSTOB - Institut Springe
Three types of D neg
RHD SMP1
SMP1
RHCE
RHCE
5’
5’
RHD SMP1 RHCE5’
RHD SMP1 RHCE5’
D+
D-
D-
D-
RHD deletion
„large“ hybrid
Stop codon, frameshift, splice site mutation ...
38DRK-Blutspendedienst NSTOB - Institut Springe
Other D negative RHD alleles
� Nonsense mutations
�RHD(W16X), RHD(W90X), RHD(Y330X)
� „Small“ deletions/insertions causing frameshift
�RHD(488del4), RHD(del711),RHD(1253instT)
� Splice site mutations (may express some D)
�RHD(IVS3+1G>A), RHD(G212V), RHD(1227G>A)
� Complex changes
�RHDΨ
�Unusual hybrid pattern, „normal“ RHD
39DRK-Blutspendedienst NSTOB - Institut Springe
Importance of C-terminal end
131 135 186 201 263 266 321 324 391
417
107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
32
11
2
72
53
75
94
Ankyrin-
interacting
residues(Nicolas V et al. 2004)
40DRK-Blutspendedienst NSTOB - Institut Springe
Shortened proteins don‘t fit the membrane
131 135 186 201 263 266 321 324 391
417
107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
32
11
2
72
53
75
94
Ankyrin-
interacting
residues(Nicolas V et al. 2004)
41DRK-Blutspendedienst NSTOB - Institut Springe
D variants
� Partial D
�Qualitative change of antigen D
�Anti-D immunization possible
�Loss of epitopes defined by monoclonal antibodies
�Antigen density normal, decreased or enhanced
�Molecular causes:
�RHD-CE-D hybrid alleles
�Single amino acid substitution in or near the extracellular loops
�Multiple dispersed amino acid substitutions
42DRK-Blutspendedienst NSTOB - Institut Springe
RhD loops and D antigen
D cat VD DBT CE
DFR D cat VI
D cat IV DHAR
Rh33Evans
Rh32DW
BARCFPTT
6
4 3
43DRK-Blutspendedienst NSTOB - Institut Springe
Single amino acid substitutions in partial D
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
44DRK-Blutspendedienst NSTOB - Institut Springe
Single amino acid substitutions in partial D
131 135 186 201 263 266 321 324 391
417
107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
32
11
2
72
53
75
94
New RhD model
45DRK-Blutspendedienst NSTOB - Institut Springe
D variants
� Partial D
�Qualitative change of antigen D
�Anti-D immunization possible
�Loss of epitopes defined by monoclonal antibodies
�Antigen density normal, decreased or enhanced
�Molecular causes:
�RHD-CE-D hybrid alleles
�Single amino acid substitution in or near the extracellular loops
�Multiple dispersed amino acid substitutions
46DRK-Blutspendedienst NSTOB - Institut Springe
RH phylogeny
Cluster of weak D type 4
alleles
Cluster of Dcategory IVa
alleles
Cluster of normal alleleRHD
Dc(W16C)e
Dce
DcEce
Ce cE
DCe
weak D type 1
DNB
D ca teg ory VI type II
wea k D typ e 2
DNU
D c ategory VI type I
Cluster of DAU
a llele
47DRK-Blutspendedienst NSTOB - Institut Springe
D variants
� Weak D and Del
�Mainly quantitative reduction of antigen D
�Usually no Anti-D immunization observed
�Molecular causes:
�Single amino acid substitution in transmembraneous and intracellular parts of the Rh protein
�Multiple dispersed amino acid substitutions
�Splice site mutations (Del)
48DRK-Blutspendedienst NSTOB - Institut Springe
Single amino acid substitutions in weak D
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
49DRK-Blutspendedienst NSTOB - Institut Springe
Single amino acid substitutions in weak D
131 135 186 201 263 266 321 324 391
417
107 158 167 230 231 282 290 347 358
Intracellular
Exofacial
32
11
2
72
53
75
94
New RhD model
50DRK-Blutspendedienst NSTOB - Institut Springe
RH Blood Group
� Rh protein structure
�Structural model
�Differences of RhD and RhCE
� Genetics
�Structure of an RH gene
�Structure of RH locus and the RHD deletion
� Prediction of the phenotype
�Molecular basis of antigen D
�Molecular basis of other main antigens
�Testing strategies, pitfalls and limitations
51DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen Cw
Arg at pos. 41
52DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen E
Pro at pos. 226
53DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen E
Pro at pos. 226
Additional mutations
may cause
„partial E“
54DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen E
RhcE(M167K): E var I, expresses Ew antigen
Additional mutations
may cause
„partial E“ Strobel et al. 2004
55DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen c
Pro at pos. 103
56DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen G
Ser at pos. 103
57DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen C
Ser at pos. 103
58DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen C
Ser at pos. 103
Overall structure must be RhCE
59DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen C
Ser at pos. 103
Cys 16 must bepresent
Overall structure must be RhCE
60DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen e
Ala at pos. 226
61DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen e
Ala at pos. 226
Overall structure must be RhCE
62DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen e
Ala at pos. 226
Overall structure must be RhCE
Exon 5 may be RHD
63DRK-Blutspendedienst NSTOB - Institut Springe
32
11
2
72 75 131 134 188 207 257 264 307 333 389
417
53 94 110 154 169 226 238 283 286 352 370
Molecular basis of antigen Ce (Rh7)
Ser at pos. 103 and Ala at pos. 226
Overall structure must be RhCE
64DRK-Blutspendedienst NSTOB - Institut Springe
Further complexities
� „African“ RHCE-variants
� -D-
� Rhnull
�Amorph type:
defect of RH
�Regulator type:
defect of RHAG
65DRK-Blutspendedienst NSTOB - Institut Springe
RH Blood Group
� Rh protein structure
�Structural model
�Differences of RhD and RhCE
� Genetics
�Structure of an RH gene
�Structure of RH locus and the RHD deletion
� Prediction of the phenotype
�Molecular basis of antigen D
�Molecular basis of other main antigens
�Testing strategies, pitfalls and limitations
66DRK-Blutspendedienst NSTOB - Institut Springe
Purpose of molecular testing
� Antigen prediction in the absence of suitable RBC
�Prenatal diagnoses
�Recently transfused patients
�Positive DAT and similar serologic problems
� Detection of weak antigens
�Donor screening
�Confirmation of serologic results
� Characterization of antigenic variants
�Discrimination weak D / partial D alleles
�Characterization of variants of other antigens
67DRK-Blutspendedienst NSTOB - Institut Springe
Testing strategies
� Direct check for antigenic polymorphisms
�Antigen Cw prediction
� Indirect check for antigenic polymorphisms
�Antigen C prediction based on intron 2
�Antigen D prediction based on intron 4
�Antigen D prediction based on non-exofacial
positions
� Exclusion of additional polymorphisms
68DRK-Blutspendedienst NSTOB - Institut Springe
Strategies for the prediction of D antigen
� RHD PCR should focus on loops 3 to 6
�Corresponds to RHD exon 4 to 7
�Absence of RHD in these loops => D negative
�Presence of RHD in all loops => likely D positive
�Presence of RHD in some loops => likely partial D
�Many partial D and hybrid-type D negative will be
typed correctly
� „Other“ changes must be detected individually
�RHDpsi, frequent stop/splice/frameshift mutations
69DRK-Blutspendedienst NSTOB - Institut Springe
A solution to antigen D prediction
B
A control
Intron 4
Exon 7
control
Intron 7 RHD(W16X) RHDΨ
RHD
neg
ativ
e
D c
at VI t
ype II
RHD
-CE(8
-9)-
D
RHD
(W16X)
RHD
Ψ
Cd
es
D c
at IV
typ
e III
sta
nd
ard
RHD
70DRK-Blutspendedienst NSTOB - Institut Springe
Exclusion of additional polymorphisms
� Which alleles must be detected?
� Alleles found as single case report?
� Alleles detected in a single region?
� Alleles detected by a single group?
� Alleles with very low frequency?
� D negative alleles
? Del alleles
? Partial D alleles
? Weak D alleles
? RHCE-alleles with unexpected D-expression (e.g. ceRT)
71DRK-Blutspendedienst NSTOB - Institut Springe
Strategies for the prediction of antigen C
� Cys at position 16
�Shared by most c-alleles of cDe (Africans!)
�Absence predicts C negative phenotype
� RHD exon 2
�Also present in RHD (e.g. cDE)
�Absence predicts C negative phenotype
� C-specific insertion in intron 2
�Best predicting polymorphism in Europeans
�Fails in Ccdes
72DRK-Blutspendedienst NSTOB - Institut Springe
Strategies for the prediction of antigen e
� Usual approach: Ala 226 in RHCE exon 5
�Fails in RHCE-D(5)-CE
�No information for RHD-CE(5)-D
�Minimal RHCE-context unknown
73DRK-Blutspendedienst NSTOB - Institut Springe
Prediction of antigen Ce (Rh7)
� Simultaneous presence of Ser 103 and Ala 226
in RHCE
�Ser 103 coded by exon 2
�Ala 226 coded by exon 5
� Two problems:
�Distance on DNA: ~ 17,000 bp
�Simultaneous testing of 3 determinants:
�Ser 103, Ala 226, RhCE
74DRK-Blutspendedienst NSTOB - Institut Springe
Additional pitfalls in Rh antigen prediction
� Regulator-type RhNull
�Normal Rh genes, no Rh antigens
� Sporadic mutations
�How many samples and regions must be tested?
� Unexplained non-expression or Rh proteins
�D negative type with anti-D despite „normal“ RHD
75DRK-Blutspendedienst NSTOB - Institut Springe
More facts --- more questions